CN101928715A

CN101928715A - Salviamiltiorrhizabge-cytochrome P450 (SmP450) gene as well as coded protein and application thereof

Info

Publication number: CN101928715A
Application number: CN 200910148593
Authority: CN
Inventors: 黄璐琦; 戴住波; 崔光红; 张夏楠
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2009-06-30
Filing date: 2009-06-30
Publication date: 2010-12-29

Abstract

The invention discloses a cytochrome P450 (SmP450) gene as well as a coded protease and application thereof. The gene is obtained by clone from salviamiltiorrhizabge by utilizing a cDNA chip technology, which fills the blank of separating and cloning a structure-modification type functional gene which is cytochrome P450 (SmP450) gene from a rare traditional Chinese medicine of salviamiltiorrhizabge. The cytochrome P450 (SmP450) gene has a nucleotide sequence shown as SEQ ID NO.1, or a homological sequence adding, substituting, inserting or lacking one or more nucleotides or allelic genes and derived nucleotide sequence thereof. The protein coded by the cytochrome P450 (SmP450) gene has an amino acid sequence shown as SEQ ID NO.2 or a homological sequence adding, substituting, inserting or lacking one or more amino acids. The cytochrome P450 (SmP450) can improve the content of tanshinone which is diterpene active element in the salviamiltiorrhizabge by the biotechnology, is beneficial to the quality improvement of the salviamiltiorrhizabge, can be used for the breeding selection and has good application prospect.

Description

The red sage root one Cytochrome P450 (SmP450) gene and encoded protein and application

Technical field

The invention belongs to biological technical field, relate generally to utilize in the cDNA chip technology clone red sage root various with its activeconstituents biosynthesizing involved enzyme gene and coded product and application, relate in particular to enzyme gene and the coded product and the application of red sage root main active ingredient phenolic acids and diterpene quinone its structural modification of participation in biosynthesizing, belong to medicinal plant genetically engineered field.

Background technology

The formation of active components in medicinal plant (secondary metabolite) is the product of peculiar gene group in the Secondary Metabolism of Plant approach.Along with extensively reaching deeply of plant functional genomics research, show unique characteristics and have the research of the synthetic correlation function gene of medicinal plant secondary metabolism of broad prospect of application to become the focus of research gradually, the clone of these genes will provide fundamental basis for the formation that the biosynthetic pathway and the regulatory mechanism thereof of parsing active components in medicinal plant are conciliate release material quality, bring wide application space for utilizing biotechnology to improve target component content or direct production effective constituent or intermediate simultaneously.

A lot of activeconstituentss such as taxol, Artemisinin, ginsenoside Rg3, camptothecine, vinealeucoblastine(VLB), Tanshinone II A, trypterygine first, second element or the like are arranged in the medicinal plant.In the biosynthetic process, the biological structure that all relates to is in various degree modified in the body of these compounds, and wherein the modification of Cytochrome P450 is very crucial.Because most of P450 are very low and unstable at the intravital content of plant, directly separation and purification P450 enzyme is very difficult, therefore clone the P450 gene, research P450 gene expression and regulation, the function by method research P450 such as heterogenous expression, deletion mutantion and mutant complementations just becomes an important channel.Biochip technology is a new and high technology that grows up the eighties in 20th century, it can be monitored simultaneously in real time to all genetic expressions of biological whole genome, as brand-new, a strong technology platform, be widely used in the research of functional genome, also become the strong means of excavating new gene.Medicinal plant red sage root Salvia miltiorrhiza Bge. is conventional Chinese medicine simply, contain abundant bioactive ingredients, wherein mainly contain two active components: there are the vestige of Cytochrome P450 modification substantially in fat-soluble diterpene quinone and water miscible phenolic acid compound on their structures.The present invention uses the Gene-Chip technology, and to cloning and analyze with the closely-related Cytochrome P450 of effective constituent in the red sage root, and the applied bioengineering technology has been carried out research and development to it in conjunction with 5 ' RACE.Before the present invention comes forth, any disclose or reported cytochrome P450 gene mentioned in the present patent application and aminoacid sequence thereof are not arranged as yet.

Summary of the invention

The object of the present invention is to provide a kind of red sage root Cytochrome P450 (SmP450) gene.

Second purpose of the present invention provides the protein of this genes encoding.

The present invention also provides recombinant vectors and the host cell that contains this gene.

Another object of the present invention is to provide the application of this gene.

Red sage root Cytochrome P450 provided by the present invention (SmP450) is one of following nucleotide sequence:

(1) has the nucleotide sequence shown in the SEQ ID NO.1;

(2) nucleotide sequence shown in the SEQ ID NO.1 add, replace, insert or delete homologous sequence or its allelotrope and the deutero-nucleotide sequence thereof of one or more Nucleotide.

The protein of this coded by said gene is red sage root Cytochrome P450 (SmP450), is one of following aminoacid sequence:

(1) has the aminoacid sequence shown in the SEQ ID NO.2;

(2) SEQ ID NO.2 add, replace, insert or delete one or more amino acid whose homologous sequences.

The recombinant vectors that contains red sage root Cytochrome P450 of the present invention (SmP450) gene complete sequence or partial sequence, as prokaryotic expression class carrier, eucaryon class expression vector and RNAi carrier all belong to protection scope of the present invention.

Contain the host cell of red sage root Cytochrome P450 of the present invention (SmP450) gene complete sequence or partial sequence, as the host cell that contains above-mentioned recombinant vectors also belongs to protection scope of the present invention.

Described host cell is valuable living materials.

The application of red sage root Cytochrome P450 of the present invention (SmP450) gene comprises and uses described recombinant vectors, crosses expression vector or interference carrier plant transformed cell as plant; Perhaps cultivate altogether with relevant living materials, obtain genetically modified plant rooting system and transfer-gen plant with the described Agrobacterium that contains recombinant vectors; Perhaps use described red sage root farnesyl pyrophosphate synthase (SmFPS) gene complete sequence or partial sequence to transform and obtain transgenic organism.

The notion particular content that relates in the technical solution of the present invention is as follows:

The dna molecular of said red sage root Cytochrome P450 (SmP450) gene comprises: coding has the nucleotide sequence of the active polypeptide of red sage root Cytochrome P450 (SmP450), and shows at least 70% homology from the nucleotides sequence of Nucleotide 1-1512 position among described nucleotide sequence and the SEQ ID NO.1; Perhaps described nucleotide sequence can be under 40-55 ℃ of condition with SEQ ID NO.1 in from the nucleotide sequence hybridization of Nucleotide 1-1512.Preferably, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQID NO.2.More preferably, described sequence has among the SEQ ID NO.1 nucleotide sequence from Nucleotide 1-1512 position.The isolated red sage root Cytochrome P450 of the present invention (SmP450) gene polypeptide comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.2 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.2 polypeptide of sequence.Dna molecular among the present invention comprises 8-100 continuous nucleotide in the described dna molecular.In the present invention, " isolating ", " purifying " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.

Term among the present invention " red sage root Cytochrome P450 (SmP450) (or polypeptide) " gene refers to: coding has the nucleotide sequence of the active polypeptide of red sage root Cytochrome P450 (SmP450), as 1-1512 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 1-1512 position Nucleotide of SEQ ID NO.1 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.1, in 1-1512 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.2 of also encoding out.Also comprising can be under the rigorous condition of moderate, better under highly rigorous condition with SEQ ID NO.1 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 1-1512 position.Also comprise with SEQID NO.1 in from the homology of nucleotide sequence at least 70% of Nucleotide 1-1512 position, preferably at least 80%, more preferably at least 90%, at least 95% nucleotide sequence best.Also comprising to encode has the variant form of open reading frame sequence among the proteic SEQ ID NO.1 with natural red sage root Cytochrome P450 (SmP450) identical function.These variant forms comprise (but being not limited to): several (are generally, 1-90, preferably, 1-60, more preferably, 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and (being generally in 60, preferably is in 30 to add several at 5 ' and/or 3 ' end, more preferably being in 10, is in 5 best) Nucleotide.

Term among the present invention " red sage root Cytochrome P450 (SmP450) albumen or polypeptide " refers to: have the active SEQ ID of red sage root Cytochrome P450 (SmP450) NO.2 polypeptide of sequence.This term also comprises the variant form that has with the SEQ ID NO.2 sequence of natural red sage root Cytochrome P450 (SmP450) identical function.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises the active fragments and the reactive derivative of red sage root Cytochrome P450 (SmP450), also comprises operationally being connected in the derivative that signal peptide, promotor or ribosome bind site sequence are formed.The variant form of red sage root red sage root Cytochrome P450 of the present invention (SmP450) polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low rigorous condition can with the coded albumen of the DNA of red sage root Cytochrome P450 (SmP450) DNA hybridization and the polypeptide or the albumen that utilize the serum of red sage root Cytochrome P450 (SmP450) polypeptide to obtain.

Red sage root Cytochrome P450 (SmP450) conservative property variation polypeptide refers among the present invention: compare with the aminoacid sequence of SEQ ID NQ.2, there are 10 at the most, preferably at the most 8, more preferably 5 amino acid similar performances or close amino acid are replaced and are formed polypeptide at the most.These conservative propertys variation polypeptide can be according to table 1, replaces and produces.

The present invention also comprises the analogue of red sage root Cytochrome P450 (SmP450) or polypeptide.The difference of these analogues and natural red sage root Cytochrome P450 (SmP450) polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.Described modification (not changing primary structure usually) form comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its proteolysis performance or optimized solubility property by modifying.

In the present invention, can select various carrier known in the art for use, the carrier as commercially available comprises plasmid, clay etc.

Table 1: the replacement residue in the conservative property variation polypeptide

Initial residue	Representational replacement residue	The preferred residue that replaces
			Ala(A)?	?Val；Leu；IIe?	Val?
Arg(R)?	?Lys；GIn；Asn?	Lys?
			Asn(N)?	?Gln；His；Lys；Arg?	Gln?
Asp(D)?	?Glu?	Glu?
			Cys(C)?	?Ser?	Ser?
Gln(Q)?	?Asn?	Asn?
			Glu(E)?	?Asp?	Asp?
Gly(G)?	?Pro；Ala?	Ala?
			His(H)?	?Asn；Gln；Lys；Arg?	Arg?
Ile(I)?	?Leu；Val；Met；Ala；Phe?	Leu?

Leu(L)?	?Ile；Val；Met；Ala；Phe?	Leu?
			Lys(K)?	?Arg；Gln；Asn?	Arg?

Met(M)?	?Leu；Phe；Ile?	Leu?
			Phe(F)?	?Leu；Val；Ile；Ala；Tyr?	Leu?
Pro(P)?	?Ala?	Ala?
			Ser(S)?	?Thr?	Thr?
Thr(T)?	?Ser?	Ser?
			Trp(W)?	?Tyr；Phe?	Tyr?
Tyr(Y)?	?Trp；Phe；Thr；Ser?	Phe?
			Val(V)?	?Ile；Leu；Met；Phe；Ala?	Leu?

When producing the former enzyme polypeptide of red sage root Cytochrome P450 of the present invention (SmP450), the nucleotide sequence of red sage root Cytochrome P450 (SmP450) gene operationally can be connected in expression regulation sequence, thereby form red sage root Cytochrome P450 (SmP450) expression vector.Described " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjacent, then means in reading frame adjacent for the secretion leader sequence.Host cell is prokaryotic cell prokaryocyte or eukaryotic cell among the present invention.Prokaryotic host cell commonly used comprises intestinal bacteria; Eukaryotic host cell commonly used comprises yeast cell, tobacco cell and other vegetable cell.

Existence and the quantity of rna transcription thing in cell of red sage root Cytochrome P450 (SmP450) is promptly analyzed in the expression of the also available Northern blotting of the present invention technical Analysis red sage root Cytochrome P450 (SmP450) gene product.

In addition, the nucleic acid molecule that can be used as probe among the present invention has 8-100 continuous nucleotide of red sage root Cytochrome P450 (SmP450) nucleotide coding sequence usually, preferably has 15-50 continuous nucleotide.This probe can be used for whether existing in the test sample nucleic acid molecule of coding red sage root Cytochrome P450 (SmP450).The present invention relates to whether exist in the test sample method of red sage root Cytochrome P450 (SmP450) nucleotide sequence, it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to red sage root Cytochrome P450 (SmP450) nucleotide coding sequence, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 Nucleotide.In addition, according to red sage root Cytochrome P450 (SmP450) nucleotide sequence of the present invention and aminoacid sequence, can be on the homology basis of nucleic acid homology or marking protein, screening red sage root Cytochrome P450 (SmP450) source gene or homologous protein.

In order to obtain the dot matrix of the red sage root cDNAs relevant with red sage root Cytochrome P450 (SmP450), can screen red sage root cDNA library with dna probe, these probes are under low rigorous condition, use ³²P red sage root Cytochrome P450 (SmP450) gene all or part of cooked the radioactivity mark and.The cDNA library that most is suitable for screening is the library from the red sage root.Structure is that biology field is well-known from the method in the cDNA library of interested cell or tissue.In addition, many such cDNA libraries also can buy, for example available from Clontech, and Stratagene, Palo Alto, Cal..This screening method can be discerned the nucleotide sequence of the gene family of red sage root Cytochrome P450 (SmP450).

The Nucleotide full length sequence of the gene family of red sage root Cytochrome P450 of the present invention (SmP450) or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.Except producing with recombination method, the also available solid phase technique of the proteic fragment of the present invention is produced (people such as Stewart, (1969) Solid-Phase Peptide Synthesis, WHFreeman Co., San Francisco by direct peptide synthesis; Merrifield J. (1963) J.Am Chem.Soc85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can (Foster City CA) synthesizes peptide automatically with the 431A type peptide synthesizer of Applied Biosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, be connected to produce the molecule of total length with chemical process then.Utilize red sage root Cytochrome P450 of the present invention (SmP450),, can filter out with red sage root Cytochrome P450 (SmP450) interactional material takes place, perhaps acceptor, inhibitor or antagonist etc. by various conventional screening methods.

Red sage root Cytochrome P450 provided by the present invention (SmP450) gene is to clone preparation first from the red sage root, has filled up from the blank of China's medicine plant red sage root Cytochrome P450 (SmP450) gene.Utilize the present invention can improve the content of terpene activeconstituents tanshinone material in the plants such as the red sage root by genetic engineering technique, transgene result shows that red sage root red sage root Cytochrome P450 (SmP450) gene pairs promotes the raising of tanshinone content that obvious effect is arranged.Red sage root Cytochrome P450 (SmP450) gene can be used to improve TANSHINONES Study on content and industrialization by transgenic technology, especially the quality-improving that can be used for the Chinese medicinal materials red sage root, can alleviate the weary problem of the serious plaque in tanshinone medicine source, have promoter action preferably to improving tanshinone material output, have good application prospects.

Description of drawings:

The functional domain prediction (deriving from ncbi database) of Fig. 1: SmP450;

Fig. 2: YE+Ag ⁺After handling Hairy Root Cultures of Salvia miltiorrhiza, SmP450RNA relative content and Cryptotanshinone and Tanshinone II A increase multiple;

Fig. 3: SmP450 transgene carrier;

Fig. 4: SmP450 transgenosis hairly root port section callus GFP checking;

Fig. 5: four tanshinone main components of SmP450 transgenosis hairly root content analysis.

Embodiment

The making of embodiment 1, red sage root cDNA chip

1, the separation of the total RNA of the red sage root and detection

Get Shanglou, Shaanxi red sage root (Salvia Miltiorrhiza Bge) root 2g, with the quick grind into powder of liquid nitrogen, the 10mL that is transferred to 65 ℃ of preheatings fast extracts (CTAB (W/V) 2%, Tris-HCl (pH8.0) 100mmolL in the damping fluid in mortar ^-1, EDTA 25m molL ^-1, NaCl 2.0molL ^-1, PVP402%, spermidine 0.5g/L, mercaptoethanol 2%), mixing fully vibrates; With equal-volume chloroform extracting twice, centrifugal 15 minutes of 7500g.Supernatant liquor adds the 10M LiCl of 1/4 volume, places 4 ℃ of precipitations behind the mixing and spends the night; Centrifugal 20 minutes of 7500g, (SDS 0.5%, NaCl 1molL with 500 μ L SSTE for precipitation ^-1, Tris-HCl (pH8.0) 10mmolL ^-1, EDTA 1mmolL ^-1, 65 ℃ of dissolvings 5 minutes.With the extracting of equal-volume chloroform, centrifugal 5 minutes of 13000g; Supernatant liquor adds 2 times of volume dehydrated alcohols, places 2h for-70 ℃; Centrifugal 20 minutes of 4 ℃ of 13000g, the precipitation drying at room temperature is dissolved in after 10 minutes in the water that 100 μ L DEPC handle, and detects the integrity of RNA with 1.0% agarose electrophoresis, with GenQuant nucleic acid quantification instrument mensuration A260, A280 ratio and concentration.Place-80 ℃ of refrigerators standby.

2, the structure in cDNA library

Adopt mRNA purification kit (QuichprepTM Micro mRNA PurifiCation Kit, Pharmacia company) behind the separating mRNA, by add EcoR I/Not I joint at cDNA molecule two ends, phosphorylation under the effect of T4 polynucleotide kinase, with expression vector λ ZAP Express Predigested Vector (ZAP Express Predigested VectorKit, stratagene company) connects, adopt the ZAP Express Pridigested Gigapack CloningKits (EcoRI/CIAP-Treated) of stratagene company that packaging protein is connected product in external packing then, infect E.coli XL1-Blue MRF ' and be built into the cDNA library.

3, the preparation of cDNA chip

3.1PCR amplification

Picking separates good plaque and is dissolved in the 100 μ L SM damping fluids (Amresco company) behind the mixing as pcr template.The pcr amplification primer is the partial sequence of λ ZAP Express multiple clone site both sides: the M13-20 primer: 5 '-GTAAAACGACGGCCAGTG-3 ', the BK reverse primer: 5 '-GGAAACAGCTATGACCTTG-3 '.96 orifice plate PCR reaction systems are prepared following mixture: dNTP (25mmolL ^-1), 600 μ L; Mg2 ⁺600 μ L, 10 * damping fluid, 1000 μ L; Taq (Takara Ex Taq, 5U μ L ^-1), 40 μ L; M13-20 primer (12.5 μ molL ^-1), 200 μ L; BK reverse primer (12.5 μ molL ^-1) 200 μ L; High purity water complements to 10mL.After mixing, be filled to 96 hole PCR plates with volley of rifle fire branch.Respectively add 6 μ L dna profilings then, mix.In the reaction of the enterprising performing PCR of ABI9700 type gene-amplificative instrament, reaction conditions is 94 ℃ of pre-sex change 3 minutes, carries out 35 circulations then, is 94 ℃ of 30s, 54 ℃ of 30s, 72 ℃ 2 minutes, extended 5 minutes after 72 ℃ after the loop ends.Get 3 μ L reaction product and containing electrophoresis on 1.5% the sepharose of EB, under SYNGENE type gel imaging system, observe, take pictures.

3.2PCR product purification

Use 96 hole Multiscreen filter plates (Millipore company) purifying plates to carry out the purifying of PCR product.The sepharose detection is changed in the purifying plate for the PCR product of single band (100 μ L); The purifying plate is put on the vacuum unit of Millipore, in 15mmHg vacuum filtration 10 minutes, until draining; Add 100 μ L water, 15mmHg vacuum filtration 10 minutes is until draining; Take off PCR purifying plate from the vacuum filtration device, add the water of 40 μ L (about pH8.0), the purifying plate is put 20～30 minutes resuspended DNA of jog to the shaking table; With the purified product sucking-off, place and measure concentration and purity on the enzyme plate; Get 1 μ L purified product electrophoresis detection on 1.5% sepharose; With purified product vacuum-drying.

3.3cDNA chip point system

Concentration difference according to said determination adds an amount of 50%DMSO as sampling liquid, regulates concentration to about 250ng μ L ^-1After, will prepare chip point system in sample transfer to 384 orifice plate.Point sample instrument is the OmniGrid of GeneMachine company 100.The point sample parameter is: pin: 2 * 12, and X:8000, Y:6000; Dot spacing: 300microns; Dot matrix: 13 * 14; Spacing of lattice: 500microns; Matrix pattern: 4 * 12.

Embodiment 2: the clone of red sage root SmP450 gene

1, chip hybridization analysis

Hairy Root Cultures of Salvia miltiorrhiza is that the mode of Agrobacterium rhizogenes 15834 direct infections is carried out Ri-plasmid conversion inductive hairly root.Get well-grown Hairy Root Cultures of Salvia miltiorrhiza (each 0.1g) succeeding transfer culture in 6, on the 7V solid medium, place 25 ℃ of incubators secretly to cultivate, multiple respectively at 30 days, 45 days, 60 days results.30 days materials as reference, are hybridized with the material of 45 days and 60 days respectively, repeat twice, every group is all adopted positive back mark to eliminate the error of dyestuff.

Adopt the indirect labelling method to carry out probe mark, comprise double-stranded cDNA is synthetic, T7 mediates the synthetic cRNA of RNA in-vitro transcription, random primer reverse transcription, cDNA steps such as KLENOW enzyme labelling.

(1) double-stranded cDNA is synthetic: get the total RNA of 5g, with T7-Oligo (dT) 15,5 '-AAACGACGGCCAGTGAATGTAATACGACTCACTATAGGCGCTTTTTTTTTTTTTTT TV-3 ', V can be G, C and A, Shanghai Bo Ya Bioisystech Co., Ltd) be primer, with cDNA Synthesis Kit (TaKaRa company) synthetic double chain cDNA; Double-stranded synthetic later on QIAquick PCR PurifiCation Kit (Qiagen company) purifying.

(2) the synthetic cRNA of in-vitro transcription: double-stranded cDNA is carried out the synthetic cRNA of in-vitro transcription with T7 RiboMAX Express Large Scale RNA Production System (Promega company); Use RNeasy test kit (Qiagen company) purifying then.

(3) random primer reverse transcription: get 2g cRNA, use the SuperscriptII ThermoScript II, 200U μ L ^-1(Invitrogen company), 9 Random Primer carry out reverse transcription, reverse transcription product QIAquick PCR PurifiCation Kit (Qiagen company) purifying.

(4) cDNA KLENOW enzyme labelling: get 1g cRNA reverse transcription product, carry out the KLENOW mark with random primer, marked product is drained behind the purifying with QIAquick PCR PurifiCation Kit (Qiagen company) purifying.DATP in the labeling process, dGTP, dTTP working concentration are 120M, and the dCTP working concentration is 60M, and Cy5-dCTP, Cy3-dCTP working concentration are 40M.

Use cy3, cy5 (Amersham company) to carry out mark respectively in different samples, be dissolved in then (3 * SSC, 0.2%SDS, 5 * Denhart ' s, 25% methane amide) in the 30L hybridization solution, spend the night in 42 ℃ of hybridization.After hybridization finishes, contain 0.2%SDS earlier about 42 ℃, washed in the liquid of 2 * SSC 5 minutes, then room temperature was washed 5 minutes in 0.2 * SSC, and slide dries laggard line scanning.Use cy3, cy5 (Amersham company) to carry out mark respectively in different samples, be dissolved in then (3 * SSC, 0.2%SDS, 5 * Denhart ' s, 25% methane amide) in the 30L hybridization solution, spend the night in 42 ℃ of hybridization.After hybridization finishes, contain 0.2%SDS earlier about 42 ℃, washed in the liquid of 2 * SSC 5 minutes, then room temperature was washed 5 minutes in 0.2 * SSC, and slide dries laggard line scanning.Chip scans with LuxScan 10K/A two channels laser scanner (CapitalBio company).Adopt GenePix Pro 4.0 image analysis software (Axon Instruments company) that chip image is analyzed, picture signal is converted into numerary signal; Then the data on the chip are carried out normalization method with the Lowess method; Determine difference expression gene with the Ttest method in conjunction with the standard of twice difference at last.

2, the acquisition of differential gene and analysis

The common differential gene of twice multiple of every core assembly sheet is carried out 5 ' the unidirectional order-checking, and the est sequence of acquisition adopts Staden Pachage (gap4) (http://staden.sourceforge.net) software under the Windows system to carry out sequence pre-treatment and splicing cluster.Remove carrier, joint and inferior quality sequence and short sequence.Use BLASTX (6 December 2005:BLAST2.2.13 released; Http:// www.ncbi.nih.gov/BLAST/) est sequence after will handling carries out homology relatively with NCBI nonredundancy Protein Data Bank (non-redundant protein database) on protein level, the terminal objective point of sample showed as up-regulated gene at 60/30 day in the results of hybridization, the BlASTX note is red sage root Cytochrome P450 (SmP450) gene, called after SmP450, this fragment has terminator codon, is 3 ' end of red sage root SmP450 gene.

3、5`-RACE

Adopt SMART ^TMThe increase 5 ' end of SmP450 of RACE cDNA Amplification Kit (Clontech company) test kit is operated to specifications.Total RNA extracts with Trizol (Invitrogene company) test kit, and step sees the test kit operational manual for details.According to above-mentioned SmP450 gene gene design special primer be: GSP1:5`-gtaggcgaggacgtcctgcgccacccc-3`, carry out the terminal amplification of cDNA5 '.The PCR condition is 94 ℃ of 5min, 94 ℃ of 30s, 68 ℃ of 30s, 72 ℃ of 3min (35 circulations), 72 ℃ of 7min.Amplify the dna fragmentation of about 400bp, sepharose reclaims test kit (Takara) and reclaims the purpose fragment, press pMD19T carrier (Takara) test kit operational manual with above-mentioned fragment cloning to the pMD19-T carrier, identify positive colony and order-checking (the biological company limited of Beijing three rich polygala roots).

The part sequencing fragment result who obtains by cDNA hybridization gained est sequence and 5 '-RACE can obtain the SmP450 full length gene cDNA sequence shown in the sequence table SEQ ID NO.1, and its deduced amino acid is shown in SEQ ID NO.2.

4, Full Length cDNA Cloning and order-checking

According to the result of 5`-RACE and the sequence of known portions, adopt directly the increase 5`-RACE-Ready cDNA of 5`-RACE institute reverse transcription of the terminal primer of 5` and 3`, use LA Taq (Takara) to carry out LA-PCR method amplification SmP450 full length sequence.Primer: SmP450-F:5`-atgaaatctcacatcatggagctaaac-3`; SmP450-R:5`-tcacgcacgcaaag-gccgcttcaa-3`.Amplify about 1500bp fragment, sepharose reclaims test kit (Takara) and reclaims the purpose fragment, is cloned into as stated above (Takara) in the pMD19-T carrier, identifies positive colony and carries out two-way order-checking (the biological company limited of Beijing three rich polygala roots).

The bioinformatic analysis of embodiment 3, SmP450 gene

The length of the red sage root diterpene synthase gene full-length cDNA that the present invention relates to is 1512bp, and detailed sequence is seen the sequence 1 in the sequence table, and wherein opening code-reading frame is positioned at 1～1512bp.Red sage root full length cDNA sequence is carried out the nucleotide homology retrieval with blast program in Non-redundantGenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translation+PDB+Swissprot+Superdate+PIR database.This gene on amino acid levels with other species in P450 higher homology is arranged, the FxxGxRxCxG feature structure territory that has simultaneously.Fig. 1.

Embodiment 4, YE+Ag ⁺Handle back SmP450 genetic expression and Cryptotanshinone, Tanshinone II A analysis on Content

Utilize YE+Ag ⁺Handle Hairy Root Cultures of Salvia miltiorrhiza, the material of collecting different time points is used for the content analysis of red sage root SmP450 gene expression amount and Cryptotanshinone, Tanshinone II A.The SmP450 gene expression amount adopts fluorescence real-time quantitative PCR.Total RNA extracts with Trizol (Invitrogene company) test kit, and step sees the test kit operational manual for details.Get 1 μ g RNA template and obtain cDNA by RT test kit (Takara company) specification sheets reverse transcription.The Real-time quantitative PCR adopts ABI Prism 7000 SequenceDetection System (Applied Biosystems, USA) and SYBGREEN PCR Master Mix (AppliedBiosystems, UK) test kit, with red sage root actin gene (GenBank accession number DQ243702) (primer sequence actin-222:5 '-GGTGCCCTGAGGTCCTGTT-3 ', actin-488:5 '-AGGAACCACCGATCCAGACA-3 ') in contrast, the conclusive evidence different time all meets the requirements and the amount of cDNA all is more or less the same.The SmP450 gene primer is: SmP450F 5-gagatcccagcagggacc-3 '; SmP450R 5 '-caagtattgcagatcgtt-3 '.By 94 ℃ of for 5min, (94 ℃ of for 30sec, 72 ℃ of for 1min of 55 ℃ of for 30sec and) 40 cycles, the PCR condition of 72 ℃ of 7min increases.The result shows: through YE+Ag ⁺After handling, the SmP450 gene expression dose raises rapidly in 24h, is reduced to the control group level afterwards gradually.After the homogenization of actin internal control gene, compare with control group, handle back 24h, YE+Ag ⁺The processing treatment group is 5.6 times of contemporaneously control group, illustrates that Hairy Root Cultures of Salvia miltiorrhiza is through YE+Ag ⁺After the processing, raise rapidly in the SmP450 gene expression dose, thereby help improving the SmP450 activity, help promoting metabolism and flow to purpose product tanshinone component and flow.

Adopt HPLC to measure Cryptotanshinone and Tanshinone II A content, chromatographic condition is: Waters 2695 high performance liquid chromatographs, Waters 2996 diode-array detectors, Millennium 32 workstations, chromatogram methyl alcohol (Tianjin); Water is triple distillation water, chloroform (analytical pure), dehydrated alcohol (analytical pure).RP-Waters C18 (150mm * 3.9mm, 5 μ m) post detects wavelength: 270nm; Column temperature: 30 ℃, its moving phase condition is: flow velocity 1.0mL.min ^-1, moving phase is 0.5% acetate methanol (A) and 0.5% acetic acid water (B) linear gradient elution: 0～25min, 30%～60%A; 25～30min, 60%～65%A; 30～50min, 65%～70%A; 50～60min, 70%～80%A; 60～61min, 80%～30%A.The result shows: through YE+Ag ⁺Handle back 1d, 2d, 4d, 6d, 9d, Cryptotanshinone content is respectively 3.5,3.9,10.2,15.7 of control group, 38.5 times; Tanshinone II A content is respectively 3.2,3.1,5.2,7.4 of control group, 9.0 times; The result shows: Hairy Root Cultures of Salvia miltiorrhiza is through YE+Ag ⁺After the processing, the significant abduction delivering of red sage root SmFPS gene expression amount energy quilt, and follow Cryptotanshinone, the run-up of Tanshinone II A.Resulting as can be known red sage root SmFPS gene is the key gene of tanshinone component biosynthetic pathway, as Fig. 2.

Embodiment 5, SmP450 transgenosis root of hair hairly root tanshinone content analysis

1.Gateway cross the member of expression vector:

Primer (the SmP450-B1:GCACAAGTTTGTACAAAAAAGCAGGCTTCACC-atgaaatctcaca tcatggag of SmP450 full length gene is expanded in design; SmP450-B2:GCACCACTTTGTACAAGAAAGCTGGGTT-tcacgcacgcaaaggcc gctt).Use the high-fidelity enzyme and carry out PCR, product is cut glue reclaim.

The BP reaction

The PCR product	50-100ng(3.5uL)?
		PDONR221?	50-100ng(0.5uL)?
BP Clonase enzyme	(1.0uL mixing down gently) with preceding

A. reaction system is put 25 degree incubations 12 hours (PCR instrument),

B. add 0.5uL Proteinase K solution 37 degree incubations 10 minutes, and stopped the BP reaction.

C. the BP reaction product is cloned through DH5 α.

D. contain on 40ug/ml kan (kantlex) the LB culture plate and spend the night, PCR checking, sequence verification, upgrading grain.

The LR reaction

BP reacts plasmid

50-150ng(3.5uL)?

[0083]?

The PH7WG2D carrier	150ng(0.5uL)?
		LR Clonase enzyme	(1.0uL mixing down gently) with preceding

A. reaction system is put 25 degree incubations 12 hours (PCR instrument),

C. the BP reaction product is cloned through DH5 α.

D. contain on 50ug/ml Spe (spectinomycin) the LB culture plate and spend the night, PCR checking, sequence verification.

The gained plasmid, upgrading grain and the method that transforms by electric shock change in the Agrobacterium then, do contrast with the empty carrier of pounding out virus gene simultaneously, as Fig. 3.

2.ACCC10060 mediated gene transforms

1. explant is cultivated in advance:

Choose 10 days red sage root aseptic seedling, blade is cut into the fritter of 0.5 * 0.5cm 2, and with blade blade surface is scratched, the mode that makes progress with the back side places blade on the MS solid medium, the pre-cultivation 2 days under the condition of the dark 8h of 25 ℃ of left and right sides illumination 16h/.

3. the activation of Agrobacterium amplification:

Picking list bacterium colony is on the new YEB substratum that contains 50mg/L Rif and 100mg/L spectinomycin on the flat board of 4 ℃ of preservations, line, 20 ℃ of overnight incubation (activation), picking list colony inoculation contains in the liquid nutrient medium of YEB of 50mg/L Rif and 100mg/L spectinomycin in 15ml, about in 28 ℃ of shaking culture to OD2600.5 (the about 23h of 250rpm), bacterium liquid is poured in the aseptic centrifuge tube, the centrifugal 10min of 4000rpm, abandon supernatant liquor, with the resuspended precipitation thalline of isopyknic MS liquid nutrient medium, be used for contaminating.

4, dip-dye and cultivation altogether:

Pre-cultivated material is put into the resuspended liquid of MS, contaminate 10min, take out explant, inhale with aseptic filter paper and remove bacterium liquid, put on the new MS solid medium, cultivate 48-72h in the dark altogether.

5, select to cultivate:

With explant sterile water wash 3 times of cultivating altogether, about 400mg/L cef water logging bubble 5min, sterile water wash 1 time, suck dry moisture then moves on the MS solid medium that contains Hgy (Totomycin) 2.5mg/L and 400mg/L cef, in dark, carry out screening and culturing, per 10 days replacing one subcultures.Select growth to grow to the resistance hairly root of 2.0cm～3.0cm rapidly, it is downcut, label changes on the MS solid medium of 2.5mg/L Hgy and 400mg/L cef separately, the positive hairly root after the degerming is changed over to the MS substratum subculture that contains 2.5mg/LHgy.

5, tanshinone assay:

6, select to cultivate:

Hairly root detects through special primer PCR through green fluorescence microscopic examination (GFP) again, and selecting positive strain is to carry out amplification culture (6, the 7V substratum), Fig. 4.

7, tanshinone assay:

Get bright hairly root 500mg vacuum-drying, extract with MeOH, the HPLC condition is the same, measures fat soluble ingredient of red sage root content.The result shows, dihydrotanshinone in crossing the transgenosis root of hair high yield strain system of expressing the SmP450 gene, and Cryptotanshinone, Tanshinone I and the comparison of Tanshinone II A content be you can well imagine high according to component: 1.2,3.0,2.0 and 1.7 times, as Fig. 5.Therefore transgene result proves, red sage root SmP450 gene pairs promotes the raising of tanshinone content that obvious effect is arranged, and the SmP450 gene can be used for utilizing transgenic technology to improve in TANSHINONES Study on content and the industrialization, has good application prospects.

SEQUENCE?LISTING

＜110〉Institute Of Chinese Materia Medica Of China Academy of Chinese Medical Sciences

＜120〉red sage root one Cytochrome P450 (SmP450) gene and encoded protein and application

<160>2

<170>PatentIn?version?3.5

<210>1

<211>1512

<212>DNA

＜213〉red sage root Salvia miltiorrhiza Bge.

<400>1

atgaaatctc?acatcatgga?gctaaacact?tcatcaacac?tcatagctct?actttccttc 60

ctctttctct?tcatctttct?caaatatgta?acaatcacaa?aatccgcaaa?ccgctactca 120

cacatcccaa?ctcccaaaac?cctccctctc?atcggaaacc?ttcacctcat?gctcggcacc 180

gacgcccccc?accgcctctt?ccgggagctc?gccgccaagc?acggccccct?catgcacctc 240

cagctcggcg?agatccactt?catcatcgtc?tcgtccgtgg?atctcgccaa?gcaggtgctc 300

aaaatccacg?acatcaactt?cgccaaccgg?cccccggggg?tggcgcagga?cgtcctcgcc 360

tacaacatga?ccgacgtcgt?ggccgccccc?tacggcgact?actggcgctt?gctgcggaag 420

atctgcacgc?tcgagcttct?gagcacgcgg?cgcgtgcagt?ccttccgccc?cataagagaa 480

gaggagaatc?tgaatctttg?cagatacctc?gcttcctgcg?ggggatcgcc?ggcgaatctg 540

tcggagaaga?tccatctgtc?gtcgtacgac?gtgatcacga?gggcggcggt?ggggacgaga 600

acgacggggc?gcgtgatgga?gagttcggtg?atttcagaga?tttcggaggt?ggggtcggga 660

ttcatgaccg?ccgatttcta?tccttccgtc?agatcactgc?ggtggatcac?catagcgccg 720

tacaagatcc?aacacattcg?ccgcaaattg?gacaagctgt?tcgacagcat?catcgaggag 780

cataaatcaa?acagagataa?ggatgccaag?tatgaagatt?ttgtggatgt?tctccttcag 840

attcaaaaag?acgggagtat?aactactgac?aacatcaaag?cagtgctcgt?ggatgtgttc 900

agtgctggaa?ctggcacgtc?agccacagcc?acagagtggg?caatgacaga?gttgatgaaa 960

aatcctagca?cactcaccaa?ggctcaagag?gaggtaagaa?gggttttcga?cgacaaggga 1020

tacgtggacg?aagacaagtt?cgaggagctc?aaatacctca?aactgatcat?caaagagaca 1080

ctgagattcc?acccgccaac?gccgctttta?atccccagaa?tcaacacaga?gagatgcgaa 1140

atcaatggat?acgagatccc?agcagggacc?agtttgatcg?tgaatgcgtg?ggcactggga 1200

agagaccccg?agtattggaa?cgatccggag?aagtttatac?cagaaagatt?tgaggaaagc 1260

gccgttgatt?tcaaagggaa?cgatctgcaa?tacttgccct?ttggttcggg?aaggagaatg 1320

tgccccggca?taatctacgg?cctcgcgaac?gtcgagttca?ttcttgcgac?gcttctctat 1380

catttcgatt?ggaagttgcc?gaaagggatg?aaaatagatg?aattggatgt?ggtggaggca 1440

tttggctcga?gtctaaaaag?gaaaaatcct?ttgcttttga?ttcccgtttt?gaagcggcct 1500

ttgcgtgcgt?ga 1512

<210>2

<211>503

<212>PRT

＜213〉red sage root Salvia miltiorrhiza Bge.

<400>2

Met?Lys?Ser?His?Ile?Met?Glu?Leu?Asn?Thr?Ser?Ser?Thr?Leu?Ile?Ala

1 5 10 15

Leu?Leu?Ser?Phe?Leu?Phe?Leu?Phe?Ile?Phe?Leu?Lys?Tyr?Val?Thr?Ile

20 25 30

Thr?Lys?Ser?Ala?Asn?Arg?Tyr?Ser?His?Ile?Pro?Thr?Pro?Lys?Thr?Leu

35 40 45

Pro?Leu?Ile?Gly?Asn?Leu?His?Leu?Met?Leu?Gly?Thr?Asp?Ala?Pro?His

50 55 60

Arg?Leu?Phe?Arg?Glu?Leu?Ala?Ala?Lys?His?Gly?Pro?Leu?Met?His?Leu

65 70 75 80

Gln?Leu?Gly?Glu?Ile?His?Phe?Ile?Ile?Val?Ser?Ser?Val?Asp?Leu?Ala

85 90 95

Lys?Gln?Val?Leu?Lys?Ile?His?Asp?Ile?Asn?Phe?Ala?Asn?Arg?Pro?Pro

100 105 110

Gly?Val?Ala?Gln?Asp?Val?Leu?Ala?Tyr?Asn?Met?Thr?Asp?Val?Val?Ala

115 120 125

Ala?Pro?Tyr?Gly?Asp?Tyr?Trp?Arg?Leu?Leu?Arg?Lys?Ile?Cys?Thr?Leu

130 135 140

Glu?Leu?Leu?Ser?Thr?Arg?Arg?Val?Gln?Ser?Phe?Arg?Pro?Ile?Arg?Glu

145 150 155 160

Glu?Glu?Asn?Leu?Asn?Leu?Cys?Arg?Tyr?Leu?Ala?Ser?Cys?Gly?Gly?Ser

165 170 175

Pro?Ala?Asn?Leu?Ser?Glu?Lys?Ile?His?Leu?Ser?Ser?Tyr?Asp?Val?Ile

180 185 190

Thr?Arg?Ala?Ala?Val?Gly?Thr?Arg?Thr?Thr?Gly?Arg?Val?Met?Glu?Ser

195 200 205

Ser?Val?Ile?Ser?Glu?Ile?Ser?Glu?Val?Gly?Ser?Gly?Phe?Met?Thr?Ala

210 215 220

Asp?Phe?Tyr?Pro?Ser?Val?Arg?Ser?Leu?Arg?Trp?Ile?Thr?Ile?Ala?Pro

225 230 235 240

Tyr?Lys?Ile?Gln?His?Ile?Arg?Arg?Lys?Leu?Asp?Lys?Leu?Phe?Asp?Ser

245 250 255

Ile?Ile?Glu?Glu?His?Lys?Ser?Asn?Arg?Asp?Lys?Asp?Ala?Lys?Tyr?Glu

260 265 270

Asp?Phe?Val?Asp?Val?Leu?Leu?Gln?Ile?Gln?Lys?Asp?Gly?Ser?Ile?Thr

275 280 285

Thr?Asp?Asn?Ile?Lys?Ala?Val?Leu?Val?Asp?Val?Phe?Ser?Ala?Gly?Thr

290 295 300

Gly?Thr?Ser?Ala?Thr?Ala?Thr?Glu?Trp?Ala?Met?Thr?Glu?Leu?Met?Lys

305 310 315 320

Asn?Pro?Ser?Thr?Leu?Thr?Lys?Ala?Gln?Glu?Glu?Val?Arg?Arg?Val?Phe

325 330 335

Asp?Asp?Lys?Gly?Tyr?Val?Asp?Glu?Asp?Lys?Phe?Glu?Glu?Leu?Lys?Tyr

340 345 350

Leu?Lys?Leu?Ile?Ile?Lys?Glu?Thr?Leu?Arg?Phe?His?Pro?Pro?Thr?Pro

355 360 365

Leu?Leu?Ile?Pro?Arg?Ile?Asn?Thr?Glu?Arg?Cys?Glu?Ile?Asn?Gly?Tyr

370 375 380

Glu?Ile?Pro?Ala?Gly?Thr?Ser?Leu?Ile?Val?Asn?Ala?Trp?Ala?Leu?Gly

385 390 395 400

Arg?Asp?Pro?Glu?Tyr?Trp?Asn?Asp?Pro?Glu?Lys?Phe?Ile?Pro?Glu?Arg

405 410 415

Phe?Glu?Glu?Ser?Ala?Val?Asp?Phe?Lys?Gly?Asn?Asp?Leu?Gln?Tyr?Leu

420 425 430

Pro?Phe?Gly?Ser?Gly?Arg?Arg?Met?Cys?Pro?Gly?Ile?Ile?Tyr?Gly?Leu

435 440 445

Ala?Asn?Val?Glu?Phe?Ile?Leu?Ala?Thr?Leu?Leu?Tyr?His?Phe?Asp?Trp

450 455 460

Lys?Leu?Pro?Lys?Gly?Met?Lys?Ile?Asp?Glu?Leu?Asp?Val?Val?Glu?Ala

465 470 475 480

Phe?Gly?Ser?Ser?Leu?Lys?Arg?Lys?Asn?Pro?Leu?Leu?Leu?Ile?Pro?Val

485 490 495

Leu?Lys?Arg?Pro?Leu?Arg?Ala

500

Claims

1. red sage root Cytochrome P450 (SmP450) gene is characterized in that it is one of following nucleotide sequences:

(1) dna sequence dna of SEQ ID No.1;

2. red sage root Cytochrome P450 (SmP450) is characterized in that, is one of following aminoacid sequence:

(1) has the aminoacid sequence shown in the SEQ ID No.2;

3. recombinant vectors is characterized in that: contain the described red sage root Cytochrome P450 of claim 1 (SmP450) gene complete sequence or partial sequence.

4. living materials, it is characterized in that: described living materials contains the described red sage root Cytochrome P450 of claim 1 (SmP450) gene complete sequence or partial sequence.

5. the application of the described gene of claim 1 in red sage root breeding.